CN106608890A - Phosphorylated arginine analogue, synthesis method and application thereof - Google Patents
Phosphorylated arginine analogue, synthesis method and application thereof Download PDFInfo
- Publication number
- CN106608890A CN106608890A CN201510687571.1A CN201510687571A CN106608890A CN 106608890 A CN106608890 A CN 106608890A CN 201510687571 A CN201510687571 A CN 201510687571A CN 106608890 A CN106608890 A CN 106608890A
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- Prior art keywords
- antibody
- arginine
- phosphorylation
- protein
- phosphorylated
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- 238000001308 synthesis method Methods 0.000 title abstract 2
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- 230000000865 phosphorylative effect Effects 0.000 description 1
- 102000020233 phosphotransferase Human genes 0.000 description 1
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 238000001814 protein method Methods 0.000 description 1
- 238000012797 qualification Methods 0.000 description 1
- 238000003908 quality control method Methods 0.000 description 1
- 230000022532 regulation of transcription, DNA-dependent Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
- 229920005989 resin Polymers 0.000 description 1
- 238000010916 retrosynthetic analysis Methods 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 230000019491 signal transduction Effects 0.000 description 1
- 238000004088 simulation Methods 0.000 description 1
- 239000007790 solid phase Substances 0.000 description 1
- 238000010532 solid phase synthesis reaction Methods 0.000 description 1
- 230000002269 spontaneous effect Effects 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 229910052717 sulfur Inorganic materials 0.000 description 1
- 239000011593 sulfur Substances 0.000 description 1
- 239000001117 sulphuric acid Substances 0.000 description 1
- 235000011149 sulphuric acid Nutrition 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 239000010414 supernatant solution Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 230000032258 transport Effects 0.000 description 1
- 238000002525 ultrasonication Methods 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 238000001291 vacuum drying Methods 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 210000004885 white matter Anatomy 0.000 description 1
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical class OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
- C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
- C07F9/02—Phosphorus compounds
- C07F9/28—Phosphorus compounds with one or more P—C bonds
- C07F9/38—Phosphonic acids [RP(=O)(OH)2]; Thiophosphonic acids ; [RP(=X1)(X2H)2(X1, X2 are each independently O, S or Se)]
- C07F9/3804—Phosphonic acids [RP(=O)(OH)2]; Thiophosphonic acids ; [RP(=X1)(X2H)2(X1, X2 are each independently O, S or Se)] not used, see subgroups
- C07F9/3808—Acyclic saturated acids which can have further substituents on alkyl
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/06—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies from serum
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
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- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
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Abstract
The invention discloses a phosphorylated arginine analogue, a synthesis method and application thereof, and relates to phosphorylated arginine analogues. The phosphorylated arginine analogue is characterized in that a phosphoryl acetamidine structure is adopted as a main body; the phosphorylated arginine analogue can be utilized as hapten for preparing an antibody used for recognizing pArg on protein; the antibody prepared from the phosphorylated arginine analogue has specificity of recognizing pArg, and cross reactivity with other common phosphorylation modification forms in protein is low; besides, a mouse polyclonal antibody prepared from the phosphorylated arginine analogue is mainly of IgG2a type, and can be used for screening monoclonal antibodies; and the phosphorylated arginine analogue can be further utilized as an inhibitor of protein phosphorylated arginine esterase, utilized as a biochemical reagent for studying biological functions of the protein phosphorylated arginine esterase, or a lead compound for medicinal development aimed at the protein phosphorylated arginine esterase.
Description
Technical field
The present invention relates to phosphorylation arginine analog, more particularly, to a class phosphorylation arginine analog and its synthetic method with
Using.
Background technology
The reversible phosphorylation of protein is that biosphere is most universal, a kind of most important protein post-translational modification mode, is also cell
One of key mechanism of interior signal transduction[1].With generation widely studied in biology in serine, threonine and tyrosine side chain
On O- phosphorylations compare, on protein arginic phosphorylation (pArg) as a kind of novel post translational modification form, in a large number
In being present in the prokaryotes such as antibacterial[2], and be considered as participating in the intragentic transcriptional control of antibacterial[3], protein quality control and respond
The important bioprocess such as environmental stimuli[4].However, due to pArg sheets it is unstable in Antigen presentation, it is impossible to by straight
Connect immune animal and obtain effective antibody, have not been reported for the antibody of pArg in albumen, therefore to pArg biological functions
Progress is slow.In addition, pArg also have to the condition responsives such as acid, heat, under the conditions of many biochemical analysises it is unstable
Etc. special property[5].The arginic antibody of phosphorylation on development specific recognition protein, can expand scientific circles and pArg is given birth to
The understanding of thing function, promotes upstream kinases with pArg as core, the functional study of downstream esterase and corresponding drug discovery.
According to existing limited result of study, the phosphorylation arginine (pArg) of free state may participate in following important life
Thing function：1) as the ATP in invertebratess body and energy snubber storehouse.When intracellular ATP concentration is reduced, pArg can make
For anakinetomer and phosphate radical donor, directly synthesize ATP with ADP.2) as the Auto-regulator of some cell membrane ionic pumps.Certain
A little cell membrane ions transport complex be by the phosphorylation-dephosphorylation process with ATP as phosphate group donor provides energy support with
Activity regulation, because ATP can be directly synthesized by pArg, so pArg can ion transport complex on regulating cell film indirectly
Activity and function.3) intermediary's material of anaerobic metabolism energy supply is turned to as intracellular aerobic metabolism energy supply.In cellular activity
In, only when metabolism aggravation and mitochondrion can not in time produce enough ATP, pArg has just served as the synthesis of ATP and buffer pool
Function.However, because pArg itself is also synthesized by ATP, so when body is subject to larger stressed condition, the buffer capacity of pArg
Power is limited, can only meet the energy needed for part ATP synthesis.Anaerobic reaction (glycolytic cycle) is played in energy requirement peak period
The Main Function of ATP is provided, so, pArg is probably to a certain extent that aerobic metabolism energy supply turns to anaerobic metabolism energy supply
Intermediate material.4) intracellular pH value is adjusted.When animal body is subject to metabolic stress, pArg can pass through spontaneous or enzymatic
Hydrolytic process, catches and fetters intracellular H+, reduce [H] in Cell sap+And improve [P3O4]3-Concentration, improve Cell sap delay
Ability is rushed, intracellular pH value is maintained.5) pArg can also have influence on the phosphorylation abilities of body and and then affect to grow.Phosphoric acid
The reduction of change ability causes high-energy phosphate only to be sufficiently used for maintaining basic cellular activity, and hinders other activities of body
Such as g and D, such as some molluscan veliger shells will be deformed due to the reduction of high-energy phosphate.
Recently, research arginic to phosphorylation on protein has some new developments.2009, Tim Clausen had found and reflect
First protein arginine kinases-McsB in prokaryote is made, and have studied its adjusting function that may be participated in[3]；2012,
Arginic phosphate-the YwlE of first phosphorylation is found that in bacillus subtilises, and by mass-spectrometric technique, with hay
Bacillus cereuss are gram positive bacteria model, identify 121 argininephosphoric acid sites in 87 albumen[6].Due to also not having at present
Have the antibody for pArg, therefore be badly in need of setting up the method for preparing anti-pArg antibody, so promotion the field is carried out deeper into
Research.
In the world currently for sour unstable N- phosphoric acid albumen detection (such as：Phosphorylation histidine), develop and applied and detection
Antigen analog approximate in structure carries out immunity, prepares the strategy of the antibody of identification true antigenic[7-8]。
List of references：
1.Tony Hunter.Cell,2000,100,113-127.
2.Elsholz,A.K.W.,K.Turgay,et al.Proc Natl Acad Sci U S A,2012,109(19),
7451-7456.
3.Fuhrmann,J.,A.Schmidt,et al.Science,2009,324(5932),1323-1327.
4.Schmidt A.；Trentini DB.；Spiess S.；Fuhrmann J.；Ammerer G.；Mechtler K.；
Clausen T.Mol Cell Proteomics,2014,13,537-550.
5.Besant,P.G.；Attwood,P.V.；Piggott,M.J.Current Protein and Peptide
Science,2009,10,536-550
6.Elsholz,Alexander K.W.Turgay,Becher,Hecker,Michael.Gerth,
Ulf.Proc.Nati.Acad.Sci.,2012,47,10335-10337.
7.Jung-Min Kee,Bryeanna Villani,Laura R.Carpenter,and Tom W.Muir.J.Am.
Chem.Soc.,2010,132(41),14327–14329.
8.Kee,J.M.,R.C.Oslund,et al.Nature Chemical Biology.,2013.9(7):416-428.
The content of the invention
It is an object of the invention to provide a class phosphorylation arginine analog and its synthetic method and application.
The one class phosphorylation arginine analog is based on phosphoryl ethanamidine structure, the change of a class phosphorylation arginine analog
Learning structural formula is：
The synthetic route of the one class phosphorylation arginine analog (pArg analog) is as follows：
Wherein, Z group includes
R group includes
R2, R3,R4,R5,R6,R7,R8Independent hydrogen is represented respectively, and alkyl, thiazolinyl, cycloalkyl, aryl, aralkyl, heterocycle is miscellaneous
Ring cycloalkyl, or phosphate protecting groups；
X represents a kind of chemical bond or a kind of linking group, and scope includes alkene, sub- alkene, sub- alkynes, cycloolefins, cycloalkyne,
Heterocycle alkene ,-(O-CH2-CH2)n-, or-(CH2)q, and-(CH2)qIn arbitrarily-CH2Group is replaced by-O-；
N values 1～100；Q values 1～6；
Y includes H ,-OR9,-NR9R10,-SR9,-COOR9,-C(O)NR9R10,-NHJ or-C (O) Q；
R9And R10Independent hydrogen, alkyl, thiazolinyl, cycloalkyl, aryl, aralkyl, heterocycle, heterocycloall yl groups are represented respectively；
J represents a kind of amido protecting group；
Q represents a kind of carboxylic acid protective group.
The R can be represented
The R2And R3H can be represented.
The X can represent alkane linking group or-CH2-CH2-。
The Y can represent-NR9R10,-NHJ；The R9And R10H can be represented.
The R can be representedR2And R3Represent H；X representative-CH2-CH2-；Y representative-NR9R10And R9、R10
Represent H.
The present invention obtains the novel class phosphorus for being structurally similar, having no document report with natural pArg by above-mentioned synthetic method
Acidifying arginine analog (pArg analog), the class phosphorylation arginine analog can be as hapten, for making
The antibody of pArg on standby identification albumen；Using the antibody prepared by class phosphorylation arginine analog (such as pAIE), with knowledge
The specificity of other pArg, common other phosphorylation modification forms (such as pSer pThr the pTyr) cross reaction with protein
Property is low；In addition, using the Mouse Polyclonal Antibody prepared by class phosphorylation arginine analog (such as pAIE), predominantly IgG2a
Type, can be used to screen monoclonal antibody.
The one class phosphorylation arginine analog is alternatively arranged as the inhibitor of protein phosphorylation arginine esterase (such as YwlE)
(inhibitor), as the biochemical reagents of such esterase biological function of research, or the elder generation of drug development is carried out for such esterase
Lead compound.
The present invention provides a kind of preparation flow of antibody, the flow process by immune animal (for example, mammal), by repeatedly
Implement immunity with aforementioned immunogens, collect serum from immune animal afterwards, and separated in serum using biological routine techniquess
Antibody, as described in Example 2.Aforementioned immunogens can also be used for external antibody or protein-bonded screening, such as phage display library skill
Art, and the phage and antibody combined with immunogens is separated using biological routine techniquess.It is inner or in vitro to screen what is obtained
Antibody is probably monoclonal or polyclonal antibody, as shown in embodiment 2, embodiment 5.The unphosphorylated albumen of antibody nonrecognition
Or aminoacid, only recognize phosphorylation arginine residues and other phosphorylated amino acid residues are not responded to, as described in Example 4.
The present invention has synthesized a class phosphorylation arginine analog, also constitutes hapten with aforementioned phosphoryl ethanamidine structure formula (I).
The hapten can be provided with carrier conjugation, the generation and combination for anti-phosphorylation arginine antibody (anti-pArg antibody)
Immunogen.The carrier material can be albumen, protein fragments, synthesis polypeptide or semi-synthetic polypeptide, as described in Example 2.
The invention further relates to the corresponding antibodies that immunogen is produced.Antibody phosphorylation arginine (pArg) in identification albumen
While, and nonrecognition non-phosphorylating arginine (Arg), non-esterified polypeptide, the phosphorylation amino in addition to phosphorylation arginine
Acid or the polypeptide containing other phosphorylated amino acid residues, as shown in embodiment 3, embodiment 4.
Hapten involved in the present invention can also be with detectable covalent bond.Label can be selected from enzyme, can also be from luminous
Select in material, radioactive substance or its mixture.Label is a kind of peroxidase, such as horseradish peroxidase.It is luminous
Material can be bioluminescence, chemiluminescence or fluorescent material as label.
The invention provides a kind of preparation flow of antibody.The flow process is by immune animal, by using aforementioned immunogens reality repeatedly
Immunity is applied, serum is collected from immune animal afterwards, and the antibody in serum, foregoing immune are separated using biological routine techniquess
Original can also be used for external antibody or protein-bonded screening, such as phage display library technology, and using biological routine techniquess separate with
Phage and antibody that immunogens are combined.The inner or in vitro antibody for obtaining that screens is probably monoclonal or polyclonal antibody.
The unphosphorylated albumen of antibody nonrecognition or aminoacid, an identification phosphorylation arginine residues are simultaneously residual to other phosphorylated amino acids
Base is not responded to.
The stable analogs that the present invention passes through design synthesis pArg, such as (2- ((2-aminoethyl) amino) -2-iminoethyl)
Phosphonic acid (pAIE), and by by itself and carrier protein (such as keyhole limpet hemocyanin, KLH)
Be coupled, the strategy of immune animal, obtaining first can the rabbit of pArg modifications and Mus polyclonal antibody on specific recognition protein.
By enzyme immunoassay (ELISA), plaque of protein dot blot (dot-blot), protein electrophorese (SDS-PAGE), egg
Multimedia evaluations such as white matter immunoblotting (Western bloting), antibody mediated immunity typing (isotyping), it was demonstrated that：
1) it is similar that the synthetic method for being developed can obtain novel pArg being structurally similar with natural pArg, having no document report
Thing；2) the pArg analog synthesized by can be as hapten, for preparing the antibody of pArg on identification albumen；3) pArg is applied
Antibody prepared by analog (such as pAIE), the specificity with identification pArg, repaiies with other phosphorylations common in protein
Decorations form (such as pSer pThr pTyr) cross reactivity is low；4) using the mice prepared by pArg analog (such as pAIE)
Polyclonal antibody, is predominantly IgG2aType, the condition for possessing screening monoclonal antibody.
The present invention has synthesized the class stable chemical nature pArg analog similar with pArg structures by MOLECULE DESIGN, and will
Such analog and the method for carrier protein couplet immune animal, establish phosphorylation arginine antibody in preparation energy identification of protein
Method and be prepared for polyclonal antibody, be additionally related to prepared polyclonal antibody in biologies such as biological detection, protein enrichments
Application in analysis field.Phosphorylation arginine analog (the pArg based on phosphoryl ethanamidine structure of present invention synthesis
Analog), the antibody of specific recognition phosphorylation arginine (pArg) can be generated effectively as hapten.Closed in the present invention
Into phosphorylation arginine analog (pArg analog) due to there is similar structure to naturally occurring pArg, it is also possible to make
For the inhibitor (inhibitor) of protein phosphorylation arginine esterase (such as YwlE), as studying such esterase biological function
Biochemical reagents, or carry out the lead compound of drug development for such esterase.
Description of the drawings
Fig. 1 is the general structure of class phosphorylation arginine analog of the present invention.
Fig. 2 is the general synthetic routes of class phosphorylation arginine analog of the present invention.
Fig. 3 is the molecular structural formula for synthesizing arginine analog pAIE used in example and Antibody preparation.
Fig. 4 is the synthetic route chart for synthesizing arginine analog pAIE in embodiment 1.
Fig. 5 is the schematic diagram that pAIE and KLH couplings prepare immunizing antigen in embodiment 2.
Fig. 6 is the argininephosphoric acid polypeptide chromatograph preparation figure for being used for serum titer detection in embodiment 2.
Fig. 7 is the first mass spectrometric qualification figure of the argininephosphoric acid polypeptide for being used for serum titer detection in embodiment 2.
Fig. 8 is the second order mses figure of the argininephosphoric acid polypeptide for being used for serum titer detection in embodiment 2.
Fig. 9 is that pAIE is applied in embodiment 2 for hapten, the serum titer figure after immune mouse.
Figure 10 be in embodiment 3 immune serum Jing after protein A affinity purification, the ELISA of each component determines figure.
Figure 11 be in embodiment 3 in embodiment 3 immune serum Jing after protein A affinity purification, the electrophoretogram of each component.
Figure 12 is Dot blot (dot-blot) the Evaluation on specificity figure of antibody purification in embodiment 4.
Figure 13 is immune serum ELISA antibody subtype analysis charts in embodiment 5.
Specific embodiment
With reference to the accompanying drawings and examples the invention will be further described.
Unless otherwise defined, all technologies for using and scientific terms implication are identical with the general understanding of general professional technique vocabulary.
Fig. 1 is the general structure of class phosphorylation arginine analog of the present invention.
Fig. 2 is the general synthetic routes of class phosphorylation arginine analog of the present invention.
The technology path for preparing phosphorylation arginine antibody in identification of protein of the present invention is as follows：
1. by the haptenic electronics cloud structure of calculating simulation phosphorylation arginine, design synthesis phosphorylation arginine analog, such as
(Fig. 3), and by retrosynthetic analysises, design synthesis step (Fig. 4) is final to close for N- (2- ethamine) -2- phosphonic acids ethanamidines (pAIE)
Into arginine analog pAIE, concrete synthetic route is shown in embodiment 1.
2. immunogen is prepared using the method for chemical coupling, by the phosphorylation arginine analog (pAIE) and keyhole of design synthesis
Relative keyhole limpet hemocyanin albumen (KLH) is coupled by glutaraldehyde method, Jing dialysis, concentration, obtains pAIE-KLH antigens (Fig. 5).Repeatedly make
Mice or rabbit, venous blood collection are injected with pAIE-KLH antigen immunes, and uses pAIE-BSA, pArg-BSA or obtain phosphorylation
Contained antibody is the generation of pAIE hapten really in arginine peptide fragment (pArg-pep) screening antigen checking serum, and multiple immunity is simultaneously
Antibody titer is determined, after antibody titer rises to plateau, blood sampling separates antiserum (Fig. 9).
3. the pAIE-BSA that screening antibodies are used is obtained using chemical conjugation methods, the phosphorylation arginine that screening antibodies are used
Peptide fragment (pArg-pep) design obtains non-phosphorus from protein arginine kinases McsB natural substrate CtsR by solid-phase peptide synthesis
After acidifying peptide fragment (pep), external Enzymatic Phosphorylation is carried out using protein arginine kinases McsB, and Jing after chromatograph preparative separation,
Obtain pArg-pep (referring to Fig. 6～8 and table 1, table 1 provides b, y ion matching result).
Table 1
4. antiserum Jing protein As are coupled filler after purification, obtain anti-phosphorylation arginine polyclonal antibody, using the phosphoric acid of synthesis
Changing arginine peptide fragment (pArg-pep) carries out whether ELISA experiment test affinity purification components recognize phosphorylation arginine (Figure 10),
And protein electrophoresises are utilized, determine the purity of protein (Figure 11) in each affinity purification component.Using the phosphorous acidifying essence ammonia of synthesis
Acid, phosphorylation serine, phosphorylation threonine, phosphotyrosine peptide (pArg-pep, pSer-pep, pThr-pep,
PTyr-pep), by the specificity (Figure 12) of dot-blot experiment test affinity purification antibody.Finally, using mountain sheep anti mouse
Typing two resists (IgG1,IgG2a,IgG2b,IgG3, IgM), by ELISA experiment tests affinity purification antibody subtype (Figure 13).
Specific embodiment given below.
Embodiment 1：The synthesis of phosphorylation arginine analog N- (2- ethamine) -2- phosphonic acids ethanamidines (pAIE)
Overall synthetic route is as shown in Figure 4.
The synthesis of compound 1 (2- diethyl phosphonyls-thioacetamide)：
Addition cyanogen methylphosphonic acid diethylester (5.30g, 30mmol) in round-bottomed flask, triethylamine (6.05g, 60mmol), four
Butylammonium bromide (125mg), toluene (25mL).Under argon protective condition, dry hydrogen sulfide is passed through in reaction bulb
(H2S) gas, stirring reaction 5h at 10 DEG C, subsequent normal-temperature reaction 12h.After reaction terminates, in reactant liquor excess is passed through
Nitrogen, after 4 DEG C of cooling 3h, is filtrated to get yellow solid.Gained yellow solid is filtered under nitrogen protective condition, is washed with toluene
Wash, obtain product Compound 1 (2.34g, yield 37%).
1H NMR(500MHz,CDCl3):δ8.45(s,1H,NH protons)and 7.73(s,1H,NH protons),4.19
(dq, J=14.2,7.1Hz, 4H ,-OCH2CH3), 3.44 (d, J=20.7Hz, 2H ,-PCH2),1.38(dt,J
=7.1Hz, 6H ,-OCH2CH3)ppm.
13C NMR(126MHz,CDCl3):δ 198.07 (d, J=6.5Hz ,-C=S), 63.24 (d, J=6.7Hz ,-OCH2CH3),
43.05 (d, J=125.4Hz ,-PCH2), 16.30 (d, J=6.1Hz ,-OCH2CH3)ppm.
31P NMR(200MHz,CDCl3):δ21.03ppm.
ESI-MS:234.2 (theoretical values [MW+Na]+), 234.25 (measured values).
The synthesis of compound 2 (2- diethyl phosphonyl-S- methylthioacetamide iodine salt)：
Under argon protection, by being dissolved in 2.5mL acetone for compound 1 (470mg, 2.23mmol), it is stirred at room temperature down and adds
Enter iodomethane (1.26g, 8.91mmol).After reaction 30min, white solid is filtrated to get, product washs 2-3 with absolute ether
After secondary, vacuum drying obtains compound 2 (666mg, yield 83%).
1H NMR(500MHz,CDCl3):δ4.50-4.16(m,4H,-OCH2CH3), 3.96 (d, J=22.5Hz, 2H ,-PCH2),
3.04 (d, J=0.9Hz, 3H ,-SCH3),2.67(s,2H,-NH2), 1.41 (dt, J=7.0Hz, 6H ,-OCH2CH3)
ppm.
31P NMR(200MHz,CDCl3):δ15.81ppm.
The synthesis of compound 3 (N- (2- ethylamino- t-butyl formates) -2- diethyl phosphonyls-ethanamidine)：
By compound 2 (611mg, 1.73mmol) and N- tertbutyloxycarbonyls -1，2-diaminoethane (277mg, 1.73mmol)
Methanol (5mL) is dissolved in, under argon protection, reaction 3h is stirred at room temperature.Solvent is removed under reactant liquor reduced pressure, obtains white
Color solid.By solid washed with ether twice, compound 3 (628mg, yield 78%) is obtained.
1H NMR(400MHz,D2O):δ 4.16 (p, J=7.2Hz, 4H ,-OCH2CH3), 3.39 (d, J=5.8Hz, 2H,
-C(O)NHCH2CH2), 3.28 (d, J=5.0Hz ,-C (O) NHCH2CH2),1.35(s,9H,-(CH3)3C),1.28(t,
J=7.0Hz, 6H ,-OCH2CH3)ppm.(-CH2P,exchange in D2O)
13C NMR(101MHz,D2O):δ 160.21 (d, J=6.1Hz ,-C=NH), 158.15 (s ,-C=O), 81.42 (s,
-(CH3)3), C 65.01 (d, J=6.9Hz ,-OCH2CH3),42.39(s,-C(O)NHCH2CH2),37.86(s,
-C(O)NHCH2CH2),27.70(s,-(CH3)3), C 15.69 (d, J=5.7Hz ,-OCH2CH3)ppm.
31P NMR(162MHz,D2O):δ20.51ppm.
ESI-MS:339.2 (theoretical values [MW+H]+), 339.3 (measured values).
The synthesis of compound 4 (N- (2- ethamine) -2- phosphonic acids ethanamidines)
Compound 3 (370mg, 0.80mmol) is dissolved in the hydrobromic acid acetums of 10mL 33%, is stirred at room temperature under argon protection
48h.Concentrating under reduced pressure reactant mixture, to remove and obtain oily crude product after solvent.Crude product is dissolved in after the acetic acid of 2mL 0.1%, is used
Cation exchange column (model AG 50W-X8) purification after 0.1% acetic acid balance, Jing after the acetic acid of 6mL 0.1% washes away impurity, produces
Thing 500mM ammonium acetate eluting, after multiple lyophilization, obtains compound as white solid 4 (117mg, yield 81%).
1H NMR(500MHz,D2O):(t, J=5.8Hz, 2H ,-C (NH) NHCH of δ 3.642CH2), 3.28 (t, J=5.9
Hz,2H,-CNHCH2CH2), 2.90 (dd, J=20.0,6.7Hz, 2H ,-CH2P)ppm.
13C NMR(126MHz,D2O):δ 164.39 (d, J=4.2Hz ,-C=NH), 39.73 (s ,-C (NH) NHCH2CH2),
37.15(s,-C(NH)NHCH2CH2), 34.57 (d, J=117.4Hz ,-CH2P)ppm.
31P NMR(200MHz,D2O):δ10.53ppm.
ESI-MS:182.1 (theoretical values [MW+H]+), 182.0 (measured values).
Embodiment 2：It is hapten using phosphorylation arginine analog pAIE, prepares the arginic antibody of anti-phosphorylation
The chemical coupling and immunity of pAIE and carrier protein：
As shown in figure 5, pAIE and keyhole limpet hemocyanin (KLH) are dissolved separately in into coupling buffer (0.1M Na2CO3,0.15M
NaCl, pH 8.5) in and be mixed in proportion, the final concentration of 15mM of wherein pAIE, the final concentration of 2mg/mL of KLH are slowly added to
25% glutaraldehyde after incubation at room temperature 2h, adds NaBH to final concentration of 0.5%4Solid is to final concentration 10mg/mL, room temperature reaction
2h dialysed overnights in PBS 7.4, with Bradford methods protein concentration is determined.
The pAIE-KLH of preparation as immunizing antigen, by mixing breast with isopyknic Freund's complete adjuvant or incomplete Freund's adjuvant
After change, by 200 μ g/ mices/time, 400 μ g/ rabbits/time, subcutaneous multiple spot or axillary fossa are injected in the clean level mice of 6 week old
Or 6 week old New Zealand white rabbit (BALB/C).Immunization interval 14～1 days, repeat 2～it is secondary after, venous blood collection, separate serum.
The preparation of detection antigen:
Detection antigen uses the polypeptide (sequence of chemosynthesis：Ac-KRGGGGYIKIIKV-NH2), Jing protein arginine kinases (McsB)
After phosphorylation in vitro, obtain containing the arginic polypeptide (sequence of phosphorylation：Ac-KpRGGGGYIKIIKV-NH2), Jing chromatographs point
From preparation, concrete grammar is as follows.Arginine kinase McsB-His is included first6(plasmid is by Tim Clausen problems for the plasmid of gene
Group is provided) it is transferred to the e. coli bl21 comprising pLys genes (DE3) bacterial strain, picking monoclonal.In 1L LB culture medium
Cultivate under the conditions of 37 DEG C of bacterium solution, the 250rpm of middle inoculation 1mL incubated overnight to OD600=0.8.0.5mM IPTG are added,
The abduction delivering 12h under conditions of 18 DEG C, 200rpm.Thalline adds 15mL to split after 4 DEG C, 8000rpm are collected by centrifugation
Solution liquid (lysate：50mM KH2PO4, 500mM KCl, 1mM beta -mercaptoethanols, 5% glycerol), 150 μ L 100mM PMSF
Storing solution, ultrasonication 10min at 4 DEG C, 14000g centrifugation 10min, retains supernatant solution.Supernatant is passed through into nickel post
Separate, McsB is obtained Jing after imidazole gradient eluting, purity of protein Jing electrophoresis checking, protein concentration is determined with Bradford methods.Profit
With substrate polypeptide (Ac-KRGGGGYIKIIKV-NHs of the McsB for obtaining to chemosynthesis2) carry out phosphorylation in vitro reaction.Reaction
Condition is：20mM Tris (pH=8.0), 5mM MgCl2, 1mM ATP, 15 μM of McsB, 40 μM of polypeptides, 30 DEG C
Insulation 1h.
The polypeptide of phosphorylation isolates and purifies rear (A phases by HPLC：The formic acid B phases of ultra-pure water 0.1%:The ultra-pure water of 95% acetonitrile 5%
0.1% formic acid；Pillar：TC-C18：4.6um x 15cm；Flow velocity：1mL/min.) (Fig. 6), purity and decorating site divide
(Fig. 8) do not identify through first mass spectrometric (Fig. 7) and second order mses (nano LC-MS/MS).
The measure of immune serum titre：
The mice serum obtained after each immunization interval, using containing the arginic polypeptide (sequence of phosphorylation：
Ac-KpRGGGGYIKIIKV-NH2) it is envelope antigen, by doubling dilution and the method for Enzyme-linked Immunosorbent Assay (ELISA), carry out
Titer determination and evaluation (Fig. 9).Concrete ELISA conditions are shown in embodiment 3.
Embodiment 3：The purification of the anti-phosphorylation arginine polyclonal antibody of mice
With reference to/lavation buffer solution:0.15M NaCl,20mM Na2HPO4,pH 8.0。
Elution buffer:0.1M glycine,pH 2.5.
Neutralization buffer:1M Tris-HCl,pH 8.5.
Take 20 μ L protein A Agarose microbeads (50% suspension) in miniature purification column, to drain off naturally, respectively with 5 times of posts
Volume (50 μ L) ddH2O and 5 times of column volume (50 μ L) combination buffer washing.The anti-phosphorylation arginine serum of mice (10 μ L)
With post glue top is carefully added on after 9 times of (90 μ L) combination buffer dilutions, collection is flowed through, afterwards with 10 times of column volumes (100 μ L)
Lavation buffer solution washs microballon, and with the μ L of every pipe 20 washing component is received；Washed with 10 column volumes (100 μ L) elution buffer afterwards
Resin is washed, eluting component is received with the μ L of every pipe 20, the eluting component of collection is neutralized at once with the neutralization buffer of 2 μ L.
The each component (comprising non-purified components, flowing through component, scrubbed component, elution fraction) that purification is obtained is respectively with containing phosphorus
It is acidified arginic polypeptide (sequence：Ac-KpRGGGGYIKIIKV-NH2) it is envelope antigen, functional verification is carried out by ELISA
(Figure 10), and with SDS-PAGE and silver staining Purity assessment is carried out.(Figure 11)
ELISA conditions：
Coating buffer：0.032M Na2CO3,0.068M NaHCO3,pH 9.6。
Lavation buffer solution：25mM Tris pH 8.5,137mM NaCl, 2.7mM KCl, 0.1% (v/v) Tween 20.
In 96 hole elisa Plates, add polypeptide described in (1 μM) embodiment 2 with coating buffer dilution of 50 μ L (negative per hole
Control) and MALDI-PSD, room temperature concussion incubation coating 2h；Remove and washed with lavation buffer solution 1 time after solution, subsequently add per hole
Enter lavation buffer solutions of the 200 μ L containing 1%BSA, after room temperature concussion closing 1h, remove confining liquid.Loading in antibody purification procedures
Sample, flows through, and washs and eluting each component, and with lavation buffer solution 100 times are diluted, and is added in ELISA Plate, often with the μ L of every hole 100
Individual sample arranges 3 groups of parallel, room temperature concussion incubation 45min.Remove and 3 times are washed with lavation buffer solution after solution.Mountain sheep anti mouse two resists
(GAM-HRP) 5000 times are diluted with lavation buffer solution, is added by the μ L of every hole 100, room temperature concussion incubation 45min removes two and resists
Washed 3 times with lavation buffer solution after solution.The μ L of Ultra tmb substrates 100 are added per hole, after incubation at room temperature 5min 50 μ L are added
2N sulphuric acid color development stopping, determination sample and is mapped in the OD values of 450nm.(Figure 10)
Embodiment 4：The specificity characterizing method of the anti-phosphorylation arginine polyclonal antibody of mice
Lavation buffer solution：25mM Tris pH 8.5,137mM NaCl, 2.7Mm KCl, 0.1% (v/v) Tween 20
By model polypeptide pep (sequences Ac-KRGGGGYIKIIKV-NH obtained by solid phase synthesis2), phosphorylation serine polypeptide
PSer-pep (sequences Ac-KpSGGGGYIKIIKV-NH2), phosphorylation threonine polypeptide pThr-pep (sequences
Ac-KpTGGGGYIKIIKV-NH2), phosphotyrosine peptide pTyr-pep (sequences Ac-KpYGGGGYIKIIKV-NH2) and it is real
Apply phosphorylation arginine polypeptide pArg-pep (sequences Ac-KpRGGGGYIKIIKV-NH of enzyme' s catalysis in example 22) be dissolved in
PBS (pH=7.4), takes respectively 0.2 μ L (667 μM) and puts on nitrocellulose filter, and subsequent albuminous plasue is used for after natural drying
Dot blotting (dot-blot) is analyzed；Dry nitrocellulose filter is soaked in the lavation buffer solution of 1%BSA, room temperature concussion
After 1h is closed, the mice multi-resistance room temperature concussion incubation 45min with the purification of 1: 100 dilution is subsequently washed with lavation buffer solution
3 times (3 × 5min).Mountain sheep anti mouse two anti-(GAM-HRP) dilutes 5000 times with lavation buffer solution, adds nitrocellulose filter,
Room temperature concussion incubation 45min, removes and is washed with lavation buffer solution 3 times (3 × 5min) after solution.After adding ECL luminous substrate,
It is imaged with the chemiluminescence Mode scans of GE Amersham Imager 600, as a result as shown in figure 12.
Embodiment 5：The immunophenotyping of the anti-phosphorylation arginine polyclonal antibody of mice
Coating buffer：0.032M Na2CO3,0.068M NaHCO3,pH 9.6。
Lavation buffer solution：25mM Tris pH 8.5,137mM NaCl, 2.7Mm KCl, 0.1% (v/v) Tween 20.
In 96 hole elisa Plates, add per hole (1 μM) of 50 μ L coating buffer dilution pep (negative control) and
PArg-pep, room temperature concussion incubation coating 2h, removes and 1 time is washed with lavation buffer solution after solution.Remove and use washing buffer after solution
Liquid is washed 1 time, and lavation buffer solutions of the 200 μ L containing 1%BSA is subsequently added per hole, after room temperature concussion closing 1h, removes confining liquid.
The mouse resisting anteserum obtained after immunity is diluted into 100 times with lavation buffer solution, is added in 96 hole elisa Plates with the μ L of every hole 100, room
Temperature concussion incubation 45min, removes and 3 times are washed with lavation buffer solution after solution.The mountain of 100 μ L, 5000 times of dilutions is sequentially added per sky
Sheep anti-Mouse typing two resists (IgG1,IgG2a,IgG2b,IgG3, IgM), room temperature concussion incubation 45min is removed after solution with washing
Wash buffer solution 3 times.100 μ L Ultra tmb substrates are added per hole, 5min is incubated at room temperature, 50 μ L 2N sulfur are added afterwards
Sour terminate liquid, determination sample is in the OD values of 450nm, and experimental result is as shown in figure 13.
Phosphoryl ethanamidine structure Formulas I is as shown in Figure 3 in this embodiment 1.
Claims (10)
1. a class phosphorylation arginine analog, it is characterised in that based on phosphoryl ethanamidine structure, a class phosphorylation arginine
The chemical structural formula of analog is：
2. the synthetic route of class phosphorylation arginine analog as claimed in claim 1 is as follows：
Wherein, Z group includes
R group includes
R2, R3,R4,R5,R6,R7,R8Independent hydrogen is represented respectively, and alkyl, thiazolinyl, cycloalkyl, aryl, aralkyl, heterocycle is miscellaneous
Ring cycloalkyl, or phosphate protecting groups；
X represents a kind of chemical bond or a kind of linking group, and scope includes alkene, sub- alkene, sub- alkynes, cycloolefins, cycloalkyne,
Heterocycle alkene ,-(O-CH2-CH2)n-, or-(CH2)q, and-(CH2)qIn arbitrarily-CH2Group is replaced by-O-；
N values 1～100；Q values 1～6；
Y includes H ,-OR9,-NR9R10,-SR9,-COOR9,-C(O)NR9R10,-NHJ or-C (O) Q；
R9And R10Independent hydrogen, alkyl, thiazolinyl, cycloalkyl, aryl, aralkyl, heterocycle, heterocycloall yl groups are represented respectively；
J represents a kind of amido protecting group；
Q represents a kind of carboxylic acid protective group.
3. synthetic route as claimed in claim 2, it is characterised in that the R is represented
The R2And R3H can be represented；The X can represent alkane linking group or-CH2-CH2-。
4. synthetic route as claimed in claim 2, it is characterised in that the Y represents-NR9R10,-NHJ；The R9And R10Represent H.
5. synthetic route as claimed in claim 2, it is characterised in that the R is representedR2And R3Represent H；
X representative-CH2-CH2-；Y representative-NR9R10And R9、R10Represent H.
6. the application of class phosphorylation arginine analog as claimed in claim 1, it is characterised in that the class phosphorylation essence ammonia
Acid-like substance as hapten, for preparing the antibody of pArg on identification albumen.
7. in application as claimed in claim 6, it is characterised in that the antibody has the specificity of identification pArg, with protein often
Other phosphorylation modification form cross reactivities seen are low, and described other phosphorylation modification forms include pSer, pThr, pTyr；
The antibody is Mouse Polyclonal Antibody, and the Mouse Polyclonal Antibody can be IgG2aType Mouse Polyclonal Antibody, IgG2aType mice
Polyclonal antibody can be used to screen monoclonal antibody.
8. application as claimed in claim 6, it is characterised in that the class phosphorylation arginine analog is used as protein phosphorylation essence
The inhibitor (inhibitor) of propylhomoserin esterase (such as YwlE), as the biochemical reagents for studying such esterase biological function, or pin
The lead compound of drug development is carried out to such esterase.
9. application as claimed in claim 6, it is characterised in that the preparation flow of the antibody is as follows：
By immune animal, by with immunogen implementing immunity repeatedly, then serum is collected from immune animal, and using life
Thing routine techniquess separate the antibody in serum；The immunogen can be used for external antibody or protein-bonded screening, such as phage display technology
Show storehouse technology, and the phage and antibody combined with immunogens is separated using biological routine techniquess；Inner or in vitro screening
The antibody for obtaining is monoclonal or Antibodies Polyclonal antibodies；The unphosphorylated albumen of antibody nonrecognition or aminoacid, only recognize phosphorus
Acidifying arginine residues are not simultaneously responded to other phosphorylated amino acid residues；The immune animal includes but is not limited to mammal.
10. as claimed in claim 6 application, it is characterised in that the hapten and detectable covalent bond, label from
Select in enzyme, or select from luminescent substance, radioactive substance, luminescent substance and radioactive substance mixture；The label
Can be peroxidase, the peroxidase can be horseradish peroxidase.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111323283A (en) * | 2020-04-20 | 2020-06-23 | 厦门大学 | Method for enriching N-phosphorylated protein |
| CN112028934A (en) * | 2020-08-17 | 2020-12-04 | 宁波大学 | Synthesis method of phosphorylated arginine analogue |
| CN114736238A (en) * | 2022-04-14 | 2022-07-12 | 厦门大学 | Protein carboxyl phosphorylation labeling reagent containing stable isotope and preparation method and application thereof |
-
2015
- 2015-10-21 CN CN201510687571.1A patent/CN106608890A/en active Pending
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111323283A (en) * | 2020-04-20 | 2020-06-23 | 厦门大学 | Method for enriching N-phosphorylated protein |
| CN111323283B (en) * | 2020-04-20 | 2021-06-25 | 厦门大学 | Method for enriching N-phosphorylated protein |
| CN112028934A (en) * | 2020-08-17 | 2020-12-04 | 宁波大学 | Synthesis method of phosphorylated arginine analogue |
| CN112028934B (en) * | 2020-08-17 | 2022-09-27 | 宁波大学 | Synthesis method of phosphorylated arginine analogue |
| CN114736238A (en) * | 2022-04-14 | 2022-07-12 | 厦门大学 | Protein carboxyl phosphorylation labeling reagent containing stable isotope and preparation method and application thereof |
| CN114736238B (en) * | 2022-04-14 | 2024-05-24 | 广东磷基生物科技有限公司 | Protein carboxyl phosphorylation labeling reagent containing stable isotope, and preparation method and application thereof |
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